Myosin-ls are the single-headed and membrane-associated members of the myosin superfamily that are found in nearly all eukaryotic cells. Myosin-ls comprise the largest unconventional myosin family found in humans (eight genes), and the large size and expression profile of the family distinguishes it as one of the most diverse. Myosin-ls play crucial roles in regulating membrane dynamics, cytoskeletal structure, and endosomal processing. However, the details of myosin-l molecular functions are still largely unknown. A widely-expressed myosin-l isoform, myolc, has been shown to facilitate the fusion of vesicles containing glucose-transporter-4 (GLUT4) with the plasma membrane in response to insulin stimulation. This crucial step regulates the cellular uptake of glucose by controlling the number of glucose transporters present on the cell surface. Decreasing the expression of myolc via siRNA knockdowns inhibits GLUT4 movement to the plasma membrane, and over expression of myolc overrides PI3-kinase inhibitor blockage of membrane Fusion. While it is clear that insulin enhances the localization of GLUT4 vesicles with myolc and actin and away from microtubules, the molecular role of myolc in membrane fusion is not known. Our goal is to use the GLUT4 transport as a model system for determining the molecular role of myosin-l in membrane transport. We will investigate the cellular dynamics of myolc movements using high-resolution microscopy, we will investigate how myolc coordinates with microtubule motors, and we will investigate how the physical properties of myolc mediate membrane delivery. Our specific aims are as follows: 1. Dynamics and distribution of GLUT4 and the cytoskeleton. We will determine the organization and spatial kinetics of the actin and microtubule cytoskeletons during GLUT4 transport and fusion, and we will determine the specific role of myolc during this process. 2. Mechanical role of myolc in GLUT4 dynamics. We will determine the mechanical role of myolc in GLUT4 dynamics. We will determine what a protein with transport and mechano-sensing activities is required for GLUT4 movements. 3. Myolc regulation and GLUT4 recruitment. We will investigate the mechanical linkage between myolc and a putative cargo-receptor, and we will investigate myolc motor regulation.